Johann Meunier

Altered Memory Capacities and Response to Stress in p300/CBP-Associated Factor (PCAF) Histone Acetylase Knockout Mice

Chromatin remodeling by posttranslational modification of histones plays an important role in brain plasticity, including memory, response to stress and depression. The importance of H3/4 histones acetylation by CREB-binding protein (CBP) or related histone acetyltransferase, including p300, was specifically demonstrated using knockout (KO) mouse models. The physiological role of a related protein that also acts as a transcriptional coactivator with intrinsic histone acetylase activity, the p300/CBP-associated factor (PCAF), is poorly documented. We analyzed the behavioral phenotype of homozygous male and female PCAF KO mice and report a marked impact of PCAF deletion on memory processes and stress response. PCAF KO animals showed short-term memory deficits at 2 months of age, measured using spontaneous alternation, object recognition, or acquisition of a daily changing platform position in the water maze. Acquisition of a fixed platform location was delayed, but preserved, and no passive avoidance deficit was noted. No gender-related difference was observed. These deficits were associated with hippocampal alterations in pyramidal cell layer organization, basal levels of Fos immunoreactivity, and MAP kinase activation. PCAF KO mice also showed an exaggerated response to acute stress, forced swimming, and conditioned fear, associated with increased plasma corticosterone levels. Moreover, learning and memory impairments worsened at 6 and 12 months of age, when animals failed to acquire the fixed platform location in the water maze and showed passive avoidance deficits. These observations demonstrate that PCAF histone acetylase is involved lifelong in the chromatin remodeling necessary for memory formation and response to stress.

Late N-acetylcysteine treatment prevents the deficits induced in the offspring of dams exposed to an immune stress during gestation.

Prenatal infection is a major stressful experience leading to enhanced susceptibility for mental illnesses in humans. We recently reported in rats, that oxidative stress and glutathione (GSH) shortage occurred in fetal male brain after lipopolysaccharide (LPS) to the dams and that these responses might be involved in the neurodevelopmental deficits observed in adolescent offspring. Furthermore, pretreatment with N‐acetylcysteine (NAC) before LPS avoided both delayed synaptic plasticity and mnesic performance deficits. Since NAC is one of the few medications permitted in pregnant women, this study evaluated the ability of NAC to serve as a protective therapy even after the LPS challenge. Pregnant rats received a single ip injection of E. coli LPS, two days before delivery, and were given NAC in their tap water after the LPS. GSH was evaluated at the time of its expected drop in the hippocampus of male fetuses, whereas long‐term potentiation (LTP) in the CA1 area of the hippocampus and spatial memory in the water‐maze were recorded in 28‐day‐old male offspring. Post‐treatment with NAC, four hours after the LPS challenge fully prevented the drop in the GSH hippocampal content. LTP, as well as spatial learning were completely protected. NAC administration at delivery also partially restored the LTP whereas post‐treatment two days later was inefficient. Another set of dams were supplemented with α‐tocopherol prior to LPS exposure, enhancing the α‐tocopherol levels in fetal hippocampus. This treatment did not prevent the LPS‐induced synaptic plasticity impairment. These results point to fetal hippocampal GSH as a major target of the detrimental effects of in utero LPS challenge. The therapeutic window of NAC extends up to birth, suggesting that this drug might be clinically useful even after an immuno‐inflammatory episode.

Blockade of Tau Hyperphosphorylation and Aβ1–42 Generation by the Aminotetrahydrofuran Derivative ANAVEX2-73, a Mixed Muscarinic and σ1 Receptor Agonist, in a Nontransgenic Mouse Model of Alzheimer’s Disease

The main objective of the present study was to establish whether the mixed σ1/muscarinic ligand ANAVEX2-73, shown to be neuroprotective in Alzheimer’s disease (AD) models in vivo and currently in clinical phase I/IIa, could have the ability to reduce the appearance of hyperphosphorylated Tau and amyloid-β1–42 (Aβ1–42) in the Aβ25–35 mouse model of AD. We therefore first confirmed that Aβ25–35 injection induced hyperphosphorylation of Tau protein, by showing that it rapidly decreased Akt activity and activated glycogen synthase kinase-3β (GSK-3β) in the mouse hippocampus. Second, we showed that the kinase activation, and resulting Tau alteration, directly contributed to the amyloid toxicity, as co-administration of the selective GSK-3β inhibitor 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxidiazole blocked both Tau phosphorylation and Aβ25–35-induced memory impairments. Third, we analyzed the ANAVEX2-73 effect on Tau phosphorylation and activation of the related kinase pathways (Akt and GSK-3β). And fourth, we also addressed the impact of the drug on Aβ25–35-induced Aβ1–42 seeding and observed that the compound significantly blocked the increase in Aβ1–42 and C99 levels in the hippocampus, suggesting that it may alleviate amyloid load in AD models. The comparison with PRE-084, a selective and reference σ1 receptor agonist, and xanomeline, a muscarinic ligand presenting similar profile as ANAVEX2-73 on M1 and M2 subtypes, confirmed that both muscarinic and σ1 targets are involved in the ANAVEX2-73 effects. The drug, acting synergistically on both targets, but with moderate affinity, presents a promising pharmacological profile.

Interaction with 1 Protein, but not NMDA Receptor, is Involved in the Pharmacological Activity of Donepezil

In the present study, we examined the interaction of (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and σ1 receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC50 = 0.7–3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the σ1 receptor with high affinity (Ki = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5–8, 1999). Thus, we sought to determine whether an interaction with the σ1 receptor may occur in vivo under physiologically relevant conditions by evaluating the σ1 receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the σ1 receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the σ1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective σ1 receptor agonist on these behavioral responses, and an interaction of the drug with the σ1 receptor must be considered in its pharmacological actions.

Antiamnesic and Neuroprotective Effects of Donepezil against Learning Impairments Induced in Mice by Exposure to Carbon Monoxide Gas

Donepezil is a potent acetylcholinesterase inhibitor that also interacts with the σ1 receptor, an intracellular neuromodulatory protein. In the present study, we analyzed the antiamnesic and neuroprotective activities of donepezil in a mouse hypoxia model induced by repetitive CO exposure, comparing donepezils pharmacological profile with other cholinesterase inhibitors tacrine, rivastigmine, and galanthamine, and the reference σ1 agonist igmesine. CO exposure induced, after 7 days, hippocampal neurodegeneration, analyzed by Cresyl violet staining, and behavioral alterations, measured using spontaneous alternation and passive avoidance responses. When injected 20 min before the behavioral tests, i.e., 7 to 8 days after CO, all drugs showed antiamnesic properties. Preadministration of the σ1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) blocked only the igmesine and donepezil effects. The neuroprotective activity of the drugs was tested by injection 20 min before the first CO exposure (preinsult protection) or by injection 1 h after the last CO exposure (postinsult protection). All drugs alleviated the hypoxia-induced neurodegeneration and behavioral impairments when injected before CO exposure. Preadministration of BD1047 blocked both the igmesine and donepezil effects. However, when injected after CO exposure, only igmesine and donepezil induced effective neuroprotection, and the morphological and behavioral effects were BD1047-sensitive. These results showed that donepezil is a potent antiamnesic and neuroprotective compound against the neurodegeneration induced by excitotoxic insult, and its pharmacological actions as both an acetylcholinesterase inhibitor and σ1 receptor agonist contribute to its marked efficacy. In particular, the drug is a more potent postinsult protecting agent compared with more selective cholinesterase inhibitors.

Increased Amyloid- β Peptide-Induced Memory Deficits in Phospholipid Transfer Protein (PLTP) Gene Knockout Mice

Oxidative stress is recognized as one of the earliest and most intense pathological processes in Alzheimer’s disease (AD), and the antioxidant vitamin E has been shown to efficiently prevent amyloid plaque formation and neurodegeneration. Plasma phospholipid transfer protein (PLTP) has a major role in vitamin E transfers in vivo, and PLTP deficiency in mice is associated with reduced brain vitamin E levels. To determine the impact of PLTP on amyloid pathology in vivo, we analyzed the vulnerability of PLTP-deficient (PLTP-KO) mice to the toxic effects induced by intracerebroventricular injection of oligomeric amyloid-β25–35 (Aβ25–35) peptide, a non-transgenic model of AD. Under basal conditions, PLTP-KO mice showed increased cerebral oxidative stress, increased brain Aβ1–42 levels, and a lower expression of the synaptic function marker synaptophysin, as compared with wild-type mice. This PLTP-KO phenotype was associated with increased memory impairment 1 week after Aβ25–35 peptide injection. Restoration of brain vitamin E levels in PLTP-KO mice through a chronic dietary supplementation prevented Aβ25–35-induced memory deficits and reduced cerebral oxidative stress and toxicity. We conclude that PLTP, through its ability to deliver vitamin E to the brain, constitutes an endogenous neuroprotective agent. Increasing PLTP activity may offer a new way to develop neuroprotective therapies.

The γ-secretase inhibitor 2-[(1R)-1-[(4-chlorophenyl)sulfonyl](2,5-difluorophenyl) amino]ethyl-5-fluorobenzenebutanoic acid (BMS-299897) alleviates Aβ1–42 seeding and short-term memory deficits in the Aβ25–35 mouse model of Alzheimer's disease

Alzheimers disease pathomimetic toxicity could be induced in mice within one week after the intracerebroventricular (i.c.v.) injection of an aggregated preparation of the highly toxic and endogenous amyloid-β fragment Aβ(25-35). It was recently reported that Aβ(25-35) also provokes a modification of APP processing with accumulation of endogenous Aβ(1-42). We here analyzed whether a γ-secretase inhibitor, BMS-299897, attenuated this Aβ(25-35)-induced Aβ(1-42) seeding and toxicity. The compound was administered at 0.1-1 nmol/mouse, concomittantly with Aβ(25-35) (9 nmol) in male Swiss mice. After one week, the contents in Aβ(1-42) and Aβ(1-40), and the levels in lipid peroxidation were analyzed in the mouse hippocampus. Mice were submitted to spontaneous alternation, passive avoidance and object recognition to analyze their short- and long-term memory abilities. Aβ(25-35) increased Aβ(1-42) content (+240%) but failed to affect Aβ(1-40). BMS-299897 blocked the increase in Aβ(1-42) content and decreased Aβ(1-40) levels significantly. The compound did not affect Aβ(25-35)-induced increase in hippocampal lipid peroxidation. Behaviorally, BMS-299897 blocked the Aβ(25-35)-induced deficits in spontaneous alternation or novel object recognition, using a 1h intertrial time interval. BMS-299896 failed to affect the passive avoidance impairments or novel object recognition, using a 24h intertrial time interval. These results confirmed that Aβ(25-35) injection provoked an accumulation in endogenous Aβ(1-42), an effect blocked by γ-secretase inhibition. This Aβ(1-42) accumulation marginally contributed to the toxicity or long-term memory deficits. However, since the seeded Aβ(1-42) affected short-term memory, the rapid Aβ(25-35) injection Alzheimers disease model could be used to screen the activity of new secretase inhibitors.

The effects of sigma (σ1) receptor-selective ligands on muscarinic receptor antagonist-induced cognitive deficits in mice.

Cognitive deficits in patients with Alzheimers disease, Parkinsons disease, traumatic brain injury and stroke often involve alterations in cholinergic signalling. Currently available therapeutic drugs provide only symptomatic relief. Therefore, novel therapeutic strategies are needed to retard and/or arrest the progressive loss of memory.

In Vivo Characterization of ARN14140, a Memantine/Galantamine-Based Multi-Target Compound for Alzheimer’s Disease

Alzheimer’s disease (AD) is a chronic pathological condition that leads to neurodegeneration, loss of intellectual abilities, including cognition and memory, and ultimately to death. It is widely recognized that AD is a multifactorial disease, where different pathological cascades (mainly amyloid and tau) contribute to neural death and to the clinical outcome related to the disease. The currently available drugs for AD were developed according to the one-target, one-drug paradigm. In recent times, multi-target strategies have begun to play an increasingly central role in the discovery of more efficacious candidates for complex neurological conditions, including AD. In this study, we report on the in vivo pharmacological characterization of ARN14140, a new chemical entity, which was obtained through a multi-target structure-activity relationship campaign, and which showed a balanced inhibiting profile against the acetylcholinesterase enzyme and the NMDA receptor. Based on the initial promising biochemical data, ARN14140 is here studied in mice treated with the amyloidogenic fragment 25–35 of the amyloid-β peptide, a consolidated non-transgenic AD model. Sub-chronically treating animals with ARN14140 leads to a prevention of the cognitive impairment and of biomarker levels connected to neurodegeneration, demonstrating its neuroprotective potential as new AD agent.

Brain Toxicity and Inflammation Induced In Vivo in Mice by the Amyloid-β Forty-Two Inducer Aftin-4, a Roscovitine Derivative

Aftins (amyloid forty-two inducers) represent a novel class of tri-substituted purines derived from roscovitine, able to promote the generation of amyloid-β (Aβ)1-42 from amyloid-β protein precursor through γ-secretase activation in cell cultures. We here examined whether aftin-4 could provoke an amyloid-like toxicity in vivo in mice. The intracerebroventricular administration of aftin-4 (3-20 nmol) increased Aβ1-42, but not Aβ1-40, content in the mouse hippocampus, between 5 and 14 days after injection. Aftin-4 injection increased lipid peroxidation levels in the hippocampus, an index of oxidative stress. It increased brain contents in pro-inflammatory cytokines, IL-1β, IL-6, and TNFα, and GFAP immunolabeling, showing astrocytic reaction. Expression of the synaptic marker synaptophysin was decreased by aftin-4. Finally, the treatment provoked marked learning deficits, observed using different memory procedures: Spontaneous alternation in the Y-maze, place learning in the water-maze, and passive avoidance response. The systemic intraperitoneal injection of aftin-4 in the 3-30 mg/kg dose range also induced oxidative stress and learning deficits. All these alterations could be blocked by pre-treatment with the γ-secretase inhibitor BMS-299,897, confirming that the mechanism of action of aftin-4 involves secretase activity. Furthermore, we examined if the cholinesterase inhibitor donepezil and the non-steroidal anti-inflammatory drug ibuprofen could prevent aftin-4-induced memory impairments, cytokine release, and lipid peroxidation. Donepezil prevented all alterations, whereas ibuprofen prevented the increases in cytokine release and lipid peroxidation, but only marginally the memory impairments. As a whole, this study showed that in vivo injection of aftin-4 results in a rapid, acute Alzheimers disease-like toxicity in the rodent brain.